Biopharmaceuticals are the fastest growing sector within the pharmaceutical industry, with a U.S. market value of $120 billion in 2009. These proteins/peptides are mainly produced using recombinant technology and established production platforms such as microbial, yeast, or mammalian cell cultures. The effectiveness of different platforms is judged primarily on protein yield, posttranslational modifications, ease of downstream purification and the capital requirements needed for commercialization. E. coli was the first large-scale protein production host and has several advantages such as cheap fermentation runs, short generation times and high titers of recombinant protein.
Mammalian cultures (CHO cells predominantly) were introduced to overcome some of the shortfalls of the microbial expression platforms such as the formation of inclusion bodies upon high titers, difficulty in purification due to endogenous endotoxin contaminants, and most importantly microbes' lack of eukaryotic posttranslational modifications (glycosylation, acylation, disulphide bridge formation etc.) which are often required for protein folding and function. CHO cells can produce recombinant proteins with glycoprofiles similar to those of native proteins. Innovations in target-gene insertion, culture media manipulation and apoptosis inhibition have improved titers to over 5 g/L.
Currently, CHO cells are the most utilized production platform despite their high infrastructure and process costs. The rapidly growing demand for biologics of all types has caused extreme shortages in manufacturing capacity. By creating a few successful biologics, the pharmaceutical industry has heightened the public need for a greater supply of additional useful protein drugs and protein agents. The high capital requirements related with the aforementioned platforms has restricted the supply of biopharmaceuticals, prompting other production strategies to be investigated for improved economics and improved capacity.
With the advent of plant transformation technology, plants and algae have proven to be feasible bioreactors for the large-scale production of recombinant proteins. The advantages are in terms of production costs, scalability and product safety, case of storage and distribution, none of which can be matched by any current bacterial or mammalian production platform. Despite the compelling advantages, several molecular pharming initiatives have fallen short primarily due to the high costs associated with the downstream purification processes. These processes rely heavily on aqueous chromatographic technology, and can account for over 70% of the total operational costs. In addition to the operational costs, there are also issues with contamination, product degradation via proteases, and large amounts of waste produced as a byproduct of aqueous recombinant protein purification. For example, even when commercial protein is expressed in seed endosperm as a non-targeted foreign protein and left to its own devices, the protein-of-interest is often trapped in undesirable protein-protein interactions with host proteome components. See, e.g. Peters et al., Efficient recovery of recombinant proteins from cereal endosperm is affected by interaction with endogenous storage proteins, Biotechnology Journal 8, (10), 1203-1212, October 2013.
Therefore, a purification process that is cheap, clean and safe is needed to overcome the significant shortfalls found in conventional aqueous purification strategies. The invention described herein solves the limitations of current aqueous purification methods by first pinning or tethering the protein onto the surface of a cellular particle such as starch granule, a particle that is then isolated to a dry state followed by cleavage of the fusion protein employing an anhydrous method. The technology eliminates product loss due to proteolytic degradation; the dry environment prevents bacterial or pathogen contaminations, and drastically reduces the amount of environmentally harmful buffers and reagents typically used for aqueous recombinant protein purification. The novel functionalized particle bearing the protein of interest can be deployed directly or further cleaved to liberate the protein of interest, freed of its carrier domain and carrier particle.
Selective chemical cleavage has proven to be a useful way to identify proteins by observing their subsequent cleavage patterns. In 1953, there was a report of selective bond cleavages for peptides that contained serine, threonine, and glycine residues when exposed to hydrochloric acid at room temperature. The cleavages at the N-terminal of the serine and threonine followed a mechanism involving a N→O shift of hydroxyl groups. The first selective cleavage at aspartic residues was observed in 1950 when a protein was heated and incubated in a weak acid solution. This caused cleavage at aspartic and asparagine residues.
In 1993, a specific and very facile cleavable bond was observed in the gas phase. This bond was the Asp-Pro peptide bond, and is much more unstable than any other bond. The mechanism of cleavage between this peptide bond is facile due to the presence of a labile proton on the side chain of aspartic acid along with the basicity of the downstream proline. The labile proton found in the side chain of the aspartic acid is important for cleavage as its esterification inhibited cleavage. The Asp-Pro bond can be cleaved under conditions where all other peptide bonds are stable. Furthermore, the Asp-Pro pairing is amongst the rarest of all amino acid pairs found in nature. The distinct properties of the Asp-Pro bond and its rarity in peptides and proteins, makes it an ideal gas-phase cleavable linker.